Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ejn.13582 This article is protected by copyright. All rights reserved.Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer’s disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of Accepted Article This article is protected by copyright. All rights reserved. excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and supresses both excitatory and inhibitory synaptic transmission but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically -induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarization, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose -dependent manner by acting primarily through muscarinic M1 receptorsThis work was supported by the Wellcome Trust Neural Dynamics PhD programme (RTB) and the Wellcome Trust (JRM). We thank Eli Lilly and Co. for gifts of GSK -5 and M1 receptor knockout mice. We thank members of the Mellor lab for helpful discussions and J. Brown for comments on previous versions of the manuscript. The authors declare no competing financial interests

Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy

Background Triple negative breast cancers (TNBC) have poor prognoses despite aggressive treatment with cytotoxic chemotherapy. Cancer-associated fibroblasts (CAFs) are prominent in tumour stroma. Our hypothesis was that CAFs modulate chemotherapy sensitivity. Methods TNBC cells and breast fibroblasts were cultured; survival after chemotherapeutics was assessed using luciferase or clonogenic assays. Signalling was investigated using transcriptomics, reporters, recombinant proteins and blocking antibodies. Clinical relevance was investigated using immunohistochemistry. Results Breast CAFs dose-dependently protected TNBC cell lines MDA-MB-231 and MDA-MB-157, but not MDA-MB-468s, from chemotherapy. CAF-induced protection was associated with interferon (IFN) activation. CAFs were induced to express IFNβ1 by chemotherapy and TNBC co-culture, leading to paracrine activation in cancer cells. Recombinant IFNs were sufficient to protect MDA-MB-231 and MDA-MB-157 but not MDA-MB-468 cells. In TNBC patients, IFNβ1 expression in CAFs correlated with cancer cell expression of MX1, a marker of activated IFN signalling. High expression of IFNβ1 (CAFs) or MX1 (tumour cells) correlated with reduced survival after chemotherapy, especially in claudin-low tumours (which MDA-MB-231 and MDA-MB-157 cells represent). Antibodies that block IFN receptors reduced CAF-dependent chemoprotection. Conclusions CAF-induced activation of IFN signalling in claudin-low TNBCs results in chemoresistance. Inhibition of this pathway represents a novel method to improve breast cancer outcomes

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss

This is the final version of the article. Available from American Society for Clinical Investigation via the DOI in this record.The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.ABT, AC, and PMS received funding from a Wellcome Trust Collaborative Award (201529/Z/16/Z). ABT, SJB, AJB, and TMH were funded through a Medical Research Council programme leader grant provided by the MRC Toxicology Unit. CCF, LMB, AJM, and HES were funded by the Eli Lilly Company. JMB received funding through a Lilly Research Award Program (LRAP) grant (Eli Lilly). RP received funding from the Marie Curie grant “Extrabrain” (European Commission). AC is a senior principal research fellow and PMS a principal research fellow of the National Health and Medical Research Council of Australia. Tissue samples were from Randy Woltjer at the Oregon Alzheimer’s Disease Center. The Oregon Alzheimer’s Disease Center is supported by NIH grant P30AG008017

Spatial and temporal investigations of reported movements, births and deaths of cattle and pigs in Sweden

<p>Abstract</p> <p>Background</p> <p>Livestock movements can affect the spread and control of contagious diseases and new data recording systems enable analysis of these movements. The results can be used for contingency planning, modelling of disease spread and design of disease control programs.</p> <p>Methods</p> <p>Data on the Swedish cattle and pig populations during the period July 2005 until June 2006 were obtained from databases held by the Swedish Board of Agriculture. Movements of cattle and pigs were investigated from geographical and temporal perspectives, births and deaths of cattle were investigated from a temporal perspective and the geographical distribution of holdings was also investigated.</p> <p>Results</p> <p>Most movements of cattle and pigs were to holdings within 100 km, but movements up to 1200 km occurred. Consequently, the majority of movements occurred within the same county or to adjacent counties. Approximately 54% of the cattle holdings and 45% of the pig holdings did not purchase any live animals. Seasonal variations in births and deaths of cattle were identified, with peaks in spring. Cattle movements peaked in spring and autumn. The maximum number of holdings within a 3 km radius of one holding was 45 for cattle and 23 for pigs, with large variations among counties. Missing data and reporting bias (digit preference) were detected in the data.</p> <p>Conclusion</p> <p>The databases are valuable tools in contact tracing. However since movements can be reported up to a week after the event and some data are missing they cannot replace other methods in the acute phase of an outbreak. We identified long distance transports of cattle and pigs, and these findings support an implementation of a total standstill in the country in the case of an outbreak of foot-and-mouth disease. The databases contain valuable information and improvements in data quality would make them even more useful.</p

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

Second messenger-induced Ca2+-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP3), Ca2+, and cyclic ADP ribose (cADPR) that trigger Ca2+-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca2+-release from lysosomal stores. While NAADP-induced Ca2+-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca2+-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca2+-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca2+-release is unaffected by emptying ER-based Ca2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca2+-release channel

Bacterial endosymbiont Cardinium cSfur genome sequence provides insights for understanding the symbiotic relationship in Sogatella furcifera host

Background: Sogatella furcifera is a migratory pest that damages rice plants and causes severe economic losses. Due to its ability to annually migrate long distances, S.furcifera has emerged as a major pest of rice in several Asian countries. Symbiotic relationships of inherited bacteria with terrestrial arthropods have significant implications. The genus Cardinium is present in many types of arthropods, where it influences some host characteristics. We present a report of a newly # identified strain of the bacterial endosymbiont Cardinium cSfur in S. furcifera. Result: From the whole genome of S. furcifera previously sequenced by our laboratory, we assembled the whole genome sequence of Cardinium cSfur. The sequence comprised 1,103,593 bp with a GC content of 39.2%. The phylogenetic tree of the Bacteroides phylum to which Cardinium cSfur belongs suggests that Cardinium cSfur is closely related to the other strains (Cardinium cBtQ1 and cEper1) that are members of the Amoebophilaceae family. Genome comparison between the host-dependent endosymbiont including Cardinium cSfur and freeliving bacteria revealed that the endosymbiont has a smaller genome size and lower GC content, and has lost some genes related to metabolism because of its special environment, which is similar to the genome pattern observed in other insect symbionts. Cardinium cSfur has limited metabolic capability, which makes it less contributive to metabolic and biosynthetic processes in its host. From our findings, we inferred that, to compensate for its limited metabolic capability, Cardinium cSfur harbors a relatively high proportion of transport proteins, which might act as the hub between it and its host. With its acquisition of the whole operon related to biotin synthesis and glycolysis related genes through HGT event, Cardinium cSfur seems to be undergoing changes while establishing a symbiotic relationship with its host. Conclusion: A novel bacterial endosymbiont strain (Cardinium cSfur) has been discovered. A genomic analysis of the endosymbiont in S. furcifera suggests that its genome has undergone certain changes to facilitate its settlement in the host. The envisaged potential reproduction manipulative ability of the new endosymbiont strain in its S. furcifera host has vital implications in designing eco-friendly approaches to combat the insect pest

Inhibition of the inositol kinase Itpkb augments calcium signaling in lymphocytes and reveals a novel strategy to treat autoimmune disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease

Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites

Advanced gastric cancer is often accompanied by metastasis to the peritoneum, resulting in a high mortality rate. Mechanisms involved in gastric cancer metastasis have not been fully clarified because metastasis involves multiple steps and requires a combination of altered expressions of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer peritoneal dissemination. In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumour (SNU-1) and of other cell lines established from the metastasis to the peritoneal cavity (SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB). The application of a high-density cDNA microarray method made it possible to analyse the expression of approximately 21 168 genes. Our examinations of SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB showed that 24 genes were up-regulated and 17 genes down-regulated besides expression sequence tags. The analysis revealed the following altered expression such as: (a) up-regulation of CD44 (cell adhesion), keratins 7, 8, and 14 (epitherial marker), aldehyde dehydrogenase (drug metabolism), CD9 and IP3 receptor type3 (signal transduction); (b) down-regulation of IL2 receptor γ, IL4-Stat (immune response), p27 (cell cycle) and integrin β4 (adhesion) in gastric cancer cells from malignant ascites. We then analysed eight gastric cancer cell lines with Northern blot and observed preferential up-regulation and down-regulation of these selected genes in cells prone to peritoneal dissemination. Reverse transcriptase–polymerase chain reaction confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination, promise to provide a new insight into the study of human gastric cancer peritoneal dissemination